A wide variety of biological research and clinical techniques utilize synthetic nucleic acid or other nucleobase polymer probes and primers for the detection, quantification, and characterization of the genetic basis of inherited and infectious diseases. Such techniques typically rely upon hybridization of the nucleic acid probes and primers to complementary regions of DNA or RNA that characterize the disease.
Probe based assays are the basis of all studies of gene expression where selectivity for specific nucleotide species is required. Nucleic acid or other nucleobase polymer probes have long been used clinically to analyze samples for the presence of nucleic acid from infectious agents, such as bacteria, fungi, virus or other organisms, and in examining genetically-based diseases.
Nucleic acid amplification assays, using oligonucleotide primers, comprise an important class of sequence-specific detection methods used in modern biological analyses, with diverse applications in diagnosis of human disease, human identification, identification of microorganisms, paternity testing, virology, and DNA sequencing. The polymerase chain reaction (PCR) amplification method allows for the production and detection of target nucleic acid sequences with great sensitivity and specificity. PCR methods have proliferated and been adapted to form the foundation of numerous biological applications, including cloning methods, analysis of gene expression, DNA sequencing, genetic mapping, drug discovery, and numerous other applications. Methods for detecting a PCR product (i.e., an amplicon) using a nucleobase oligomer probe capable of hybridizing with the target sequence or amplicon are well known in the art.
Although such methodologies are made possible by the binding specificity of probes and primers to a nucleic acid template, the presence of polymorphic variations in a particular template may also limit the utility of specific probes, since differences between the nucleic acid sequence of the probe and the nucleic acid sequence of the template (as a result of polymorphic variation in the template) may prevent the probe from hybridizing to the template. One approach to this problem has been to design different sets of probes complementary to each polymorphic variant. The use of degenerate probe sets, however, is often technically and economically prohibitive. In addition, degenerate probe sets are ineffective in identifying unknown polymorphic variations not contemplated by the degenerate probe set.
Another approach to discriminating between nucleic acid templates having multiple polymorphic variations has been to emphasize differences attributable to one particular polymorphic site by deliberately introducing mismatches near the polymorphic site of interest using universal nucleobases that do not include hydrogen bond donor or acceptor groups (See, e.g., Margraf et al., Masking selected sequence variation by incorporating mismatches into melting probes, Hum. Mutat. 0:1-10 (2006)).
Accordingly, there is a need for improved methods and reagents for detection, quantification and characterization of nucleic acid templates having polymorphic variation.